Interactions among forest-floor guild members in
structurally simple microhabitats.

Abstract:

Intraguild predation in structurally complex habitats is thought to
weaken trophic cascades and increase food web stability. However, many
predators commonly found in leaf litter become restricted to simple
microhabitat beneath rocks and logs during periods between rains. It is
within this structurally simple microhabitat that some predators defend
rich prey resources and are likely to interact strongly as the
surrounding forest becomes too dry to forage broadly in space. We
conducted a 4-y press experiment where we removed focal predators from
unfenced field plots. To evaluate the effects of predators on one
another we removed either salamanders or centipedes from beneath
artificially placed cover objects and compared abundances of these and
other intraguild predators to those in non-removal controls. We
predicted that salamanders and centipedes would have strong negative
effects on each other and on carabid beetles and spiders. We removed a
total of 1988 salamanders and 1056 centipedes over 98 sampling dates. In
salamander removal plots spider abundance increased by 34%, and carabid
beetles decreased by 15% relative to the control. In centipede removal
plots salamanders increased by 18% and carabid beetles increased by 29%,
but spider abundance decreased by 15%. Interaction strengths were
strongest in the drier summer months when territorial predators were
confined in spatially fixed microhabitats. It is during these periods
that predators may strongly regulate the abundances of guild members. In
territorial species that defend areas beneath natural cover, the effect
of intraguild predators may be an important mechanism that regulates
distribution and abundance of forest floor predators.

Early studies of community dynamics used simple models to predict
distribution and abundance patterns of species in various systems.
Hairston and Hairston (1993) noted that simple models operated under a
number of assumptions. For example, it was assumed that links in food
chains were equal in value, and early models may have ignored the
multitude of interactions (e.g., intraguild predation (IGP) and
competition) within trophic levels that are likely to affect the
remainder of the web. More recently ecologists have begun to focus on
the kinds of biotic interactions that influence the ecology of organisms
in communities. Ecologists now understand that IGP and omnivory are
widespread (Polls and Holt, 1992; Polls, 1998; Wise and Chen, 1999;
Moya-Larano and Wise, 2007) and that it may be insufficient to look at
linear chains if we are to understand the variables that affect
community structure, stability, and ecosystem function (Polls, 1991,
1994; Bascompte, 2009). A more effective approach to understanding the
consequences of perturbations on communities is to apply network theory
which emphasizes connections within a web rather than focusing on
pair-wise interactions between species (Bascompte, 2009). Research
investigating behavior among generalist predators in food webs is
important because the strength of non-trophic interactions can influence
organisms at other positions within the web. For example, IGP is thought
to weaken trophic cascades in terrestrial habitats (Holt and Polis,
1997) and increase web stability (McCann et al., 1998) because predation
within trophic compartments should reduce pressure on more basal trophic
levels. One way to further our understanding of the consequences of
competition and/or IGP at the community level is to study the numerical
response of potentially interacting guild members to changing densities
of single predator groups in complex terrestrial webs (Moya-Lareno and
Wise, 2007).

Our research was conducted in a temperate deciduous forest-floor
system. Such systems are thought to have characteristics (e.g., high
species diversity, structural habitat complexity, IGP, and omnivory)
that should attenuate the effects of predators at more basal trophic
levels (Finke and Denno, 2004). Terrestrial salamanders, spiders,
carabid beetles, and centipedes are among the most abundant predators
living in the litter on the forest-floor at our field site. Previous
laboratory studies on local populations of ecologically similar
predators have shown that competition may be an important mechanism
influencing the distribution of secondary and tertiary consumers in the
litter of temperate forests in northeast Ohio (Gall et al., 2003,
carabid beetles; Hickerson et al., 2004, centipedes; Anthony et al.,
2007, centipedes; Figura, 2007, spiders), and a growing body of evidence
suggests that terrestrial salamanders in the family Plethodontidae are
important regulators of invertebrate communities and of decomposition of
organic material on temperate forest-floors (Burton and Likens, 1975;
Hairston, 1987; Wyman, 1998; Rooney et al., 2000; Walton, 2005; Walton
and Steckler, 2005). These salamanders can be extremely abundant (Test
and Bingham, 1948; Jaeger, 1980a; Mathis, 1991), and their biomass has
been estimated in one locality to be greater than that of birds and
equal to small mammals (Burton and Likens, 1975). The impact of this
abundant group of salamanders may exceed that estimated for forest-floor
spiders (Moulder and Reichle, 1972). Therefore, plethodontid salamanders
are expected to be influential in determining community structure in the
detrital web of temperate forests. The dominant plethodontid at our
field site is the Eastern Red-backed Salamander, Plethodon cinereus, a
territorial and completely terrestrial species (Jaeger, 1984; Mathis et
al., 1995; Petranka, 1998; Anthony et al., 2008). Natural cover objects
(e.g., rocks, bark, and logs) serve as territories for P. cinereus
during dry periods, and it is beneath such cover objects that we expect
red-backed salamanders to interact with large invertebrate predators
most strongly. The microhabitats beneath cover are generally simple in
structure as they are formed by the interface of relatively impervious
cover and the organic soil. Simple habitats generally do not support a
high diversity of predators (reviewed in Langellotto and Denno, 2004),
but in forest systems that experience periodic drying, these small
spaces serve as aggregation points for IG predators and for their
desiccation intolerant prey.

One commonly held assumption is that predation occurs within guilds
whenever adults of species "A" are large enough to eat
juveniles of species "B" and in an ontogenetic reversal (Polis
et al., 1989) when adults of species "B" prey on juveniles of
species "A." It is further assumed that the likelihood of
ontogenetic reversal of predation is high because both groups are
generalist predators that experience large changes in size through
ontogeny. Although many IG predators likely share prey resources (Wise,
1993; Foelix, 1996; Werner and Raffa, 2003), recent studies
investigating predation by adult invertebrates on juveniles of Plethodon
cinereus report no evidence of predation (Anthony et al., 2007; Figura,
2007). The full spectrum of prey preferences of most large invertebrates
are unknown due to difficulties in identifying stomach contents, (but
see Wise and Chen, 1999 for centipede predation rates on juvenile
spiders of the genus Schizocosa), but the diet of P. cinereus is well
documented (Jaeger, 1990; Maglia, 1996; Adams and Rohlf, 2000; Anthony
et al., 2008) and guild members are rarely eaten by red-backed
salamanders. These examples indicate that it may be incorrect to assume
that IGP occurs based on size asymmetries alone.

Although previous studies suggest that the relationships between
Plethodon cinereus and large arthropod predators is competitive (Gall et
al., 2003; Hickerson et al., 2004; Anthony et al., 2007; Figura, 2007)
rather than predatory, the ways in which the interactions function
within the intricate forest-floor food web are unclear. Because
terrestrial salamanders are important regulators of leaf litter
arthropods, and because other large invertebrate predators appear to be
ecologically similar, understanding both how and when these predators
interact with one another seems a logical part of determining which
mechanisms are most influential in structuring the community. For
example, Peckarsky et al. (2008) illustrate how inclusion of data on
non-predatory effects in classic textbook predator/prey data sets (e.g.,
lynx-snowshoe hair cycles) can alter our original understanding of
population and community dynamics.

Despite the growing body of literature that suggests terrestrial
salamanders are important regulators of invertebrate abundance and leaf
litter decomposition rates, there are few long-term, unenclosed field
experiments on temperate forest-floor systems that examine types and
strengths of interactions among predators that occur in specific
microhabitats beneath cover objects. Between precipitation events the
soil (humus) beneath rocks and logs retains moisture longer than the
surrounding leaf litter. Therefore, microhabitats under rocks and logs
become refugia from desiccation for terrestrial amphibians and many
arthropods (Jaeger, 1980b). During such periods, these cover objects
also become defendable territories for red-backed salamanders (Jaeger,
1972; Jaeger et al., 1982). If large invertebrate predators, like
centipedes, enter salamander territories in search of prey, they likely
interact directly with one another. Furthermore, given the structurally
simple microhabitat beneath cover, the potential for strong interactions
that affect the distribution and abundance of these guild members seems
inevitable.

We conducted a 4-y press experiment in unrestricted open field
plots to evaluate how relative predator abundances affect one another. A
press experiment is a repeated alteration of species densities (e.g.,
removals) maintained over time to examine densities of unperturbed
species. We focused our efforts on removing centipedes and salamanders
because these two taxa are ecologically similar and are numerically
abundant. We hypothesized that the removal of salamanders or centipedes
from beneath artificially placed cover objects in the field should cause
changes in the abundances of other guild members. More specifically we
predicted that salamanders and centipedes would have negative effects on
each other and on carabid beetles and spiders. These predictions are
based upon the expectation that interactions among organisms in
structurally simple habitats (i.e., beneath cover objects) should be
strong relative to those in complex habitats (i.e., leaf litter).

MATERIALS AND METHODS

On 12 and 13 Apr. 2004 we placed 216 artificial cover objects
(ACOs) on the forest-floor in the Cuyahoga Valley National Park (CVNP),
Summit County, Ohio (41[degrees]13'46.62"N,
81[degrees]31'7.77"W). The field site is mixed deciduous
forest that is dominated by Acer saccharum (Sugar Maple), Fagus
grandifolia (American Beech), Liriodendron tulipifera (Tulip Poplar),
and Quercus rubra (Red Oak) and lies on a north/northeast facing slope
(elevational range 260-271 m). We used dull white ceramic floor tiles
measuring 30 x 30 cm as ACOs, and we removed the leaf litter from
directly beneath each tile before placing the ACOs in position. This put
the tile in direct contact with the soil substrate. The ACOs were
arranged in 24 plots; each was separated by 2 m and covered a 5 x 5
[m.sup.2] area. Each of the 24 plots consisted of a cluster of nine ACOs
separated by 1 m, all of which received the same treatment application.
Each plot was randomly assigned to eight blocks, each of which contained
three treatments. The design was employed with two contstraints; (1)
adjacent plots did not receive the same treatment application, and (2)
each block was comprised of one of each treatment. The three treatments
were; (1) no removal/control (NR), (2) salamander removal (SR), and (3)
centipede removal (CR). Additional predator removal combinations would
have been interesting but would have necessitated loss of replication.
The combinations chosen were based upon previous work that suggests
individuals of the salamander, Plethodon cinereus behave aggressively
toward intraspecific intruders (reviewed in Jaeger and Forester, 1993;
Mathis et al., 1995) and centipede intruders (Hickerson et al., 2004).

Data collection began on 23 Apr. 2004 and continued through 23 May
2008. The field site was visited every 2 wk, except for winter months,
through the end of 2005, and weekly beginning in spring 2006 through the
duration of the study (total of 98 visits). Red-backed salamanders
retreat to subsurface hibernacula as deep as 1 m during Dec.-Feb. when
temperatures and precipitation are low (Grizzell, 1949; Caldwell and
Jones, 1973). During each visit we hand-turned ACOs, counted and
identified macrofauna from beneath each, and removed predators from the
appropriate treatments. All 24 plots were visited in random order on
each sampling day to remove any hourly temporal bias in sampling.
Macropredators recorded at our field site were centipedes
[Lithobiomorpha (Bothropolys and Lithobius), Scolopendromorpha
(Scolopocryptops sexspinosus) and Geophilomorpha], spiders [the largest
and most abundant were amaurobiids (Wadotes)], beetles (Carabidae; the
most abundant were Pterostichus stygicus which made up over 40% of the
total each year). Invertebrates under ACOs were not identified to finer
taxonomic resolution because identification had to be done in the field.
Invertebrates from removal treatments were hand caught and preserved in
70% ethanol. At our field site the salanaander, Plethodon cinereus, made
up 99.5% of the total number of observed salamanders. Only rarely did we
see other salamander species (P. glutinosus, Notophthalmus viridescens
and Eurycea bislineata). Salamanders in removal plots were relocated
across barriers (streams or roads) at a distance of at least 0.5 km so
that they were unable to move back into the experimental plots (Marsh et
al., 2007).

During every other visit we measured and recorded soil temperature
and percent relative humidity at the center ACO of each plot (24 points
evenly spaced throughout the site) to determine if there was variation
in the abiotic environment at the microhabitat scale that might
influence the distribution and abundance of the occupants of the
forest-floor detrital web. We used an Oakton digital max./min,
thermohygrometer to measure percent relative humidity.

STATISTICAL ANALYSES

The combined effects of treatment and season on predator abundance
were examined using multivariate analysis of covariance (MANCOVA).
Treatment and season were fixed factors (independent variables), numbers
of salamanders, centipedes, spiders, and carabid beetles were the
dependent variables, and soil temperature and relative humidity were
covariates in our model. We were unable to use repeated measures MANOVA
to analyze our data because the test assumes that conditions do not vary
from sampling date to sampling date (McCall and Appelbaum, 1973). At out
our field site, conditions vary dally and seasonally and these
conditions have strong effects on surface activity of predators. To
determine if initial abundances of predators differed among treatments,
we ran a MANOVA on the data from the first sampling day. To assess our
effectiveness in removing the centipedes and salamanders, we compared
the numbers in the control plots (NR) to the appropriate removal plots
(SR, CR). We explored differences among treatments with Scheffe's
post-hoc test which corrects alpha for all pairwise comparisons and is,
therefore, a conservative approach.

Interaction strengths were calculated to examine the effects of
each predator taxon on the other guild members. We used the raw
difference index [i.e., (N- D)/Y) where N = the number of intraguild
prey in the control, D = the number of intraguild prey in the predator
removal treatment, and Y = the abundance of predators in the removal
treatment (Berlow et al., 1999)]. For example, the effect of salamanders
on spiders was calculated by subtracting the mean numbers of spiders (D)
in the salamander removal treatment from the mean number of spiders (N)
in the control and dividing that result by the number of salamanders (Y)
in treatments where they were removed. This index is appropriate for
long term experiments that examine both positive and negative effects of
predators (Berlow et al., 1999). The mean interaction strengths were
examined over the 98 sampling days with a sign test to determine if
there were significant departures from zero. Abiotic measurements were
compared among all treatments over time. All data were log-transformed,
[log.sub.10](x + 1) to improve adherence to normality. To reduce the
probability of making a Type I error we applied Bonferroni adjustments
and considered a significant alpha to be P < 0.02.

RESULTS

EFFECTIVENESS OF TREATMENT APPLICATIONS

We removed a total of 1288 salamanders from SR and 1056 centipedes
from CR plots over the 4 y study (spanning 98 sampling dates). Initial
densities of focal predators did not differ among treatments (P = 0.106,
F = 1.82), so we conclude that our statistical findings are a direct
result of our treatment manipulations rather than resulting from
differences in initial densities in plots at our field site. We rarely
missed predators under cover objects, so our removal rate (based upon
visual inspection) approached 100% for all observed predator groups that
were encountered beneath ACOs. Despite the open plot design of our
experiment, reductions in salamander numbers persisted well after each
collection date (Fig. 1a and Fig. 2a). We reduced total salamander
abundance by 28% and adult salamander abundance by 47% in salamander
removal (SR) plots relative to control (NR) plots. Centipedes re-invaded
cover objects from the surrounding litter matrix most rapidly and by the
following collection date had reached 93% of their original numbers
(Fig. lb). We were only able to reduce centipede numbers effectively
during the spring season (Fig. 2b).

INTRAGUILD PREDATOR INTERACTIONS

We found a significant effect of treatment (P < 0.0001, F =
17.47) and season (P < 0.0001, F = 56.63) on the abundance of the
focal predators in our plots. We also found a weak treatment x season
interaction (P = 0.049, F = 1.65) that was only evident for salamanders
after the addition of soil temperature and relative humidity as
covariates in our model (Table 1). This interaction is a result of a
weak treatment effect during the summer months when salamanders retreat
from the surface (Fig. 2a). We found a strong treatment effect on
salamander, spider, and carabid beetle abundances but only a weak
treatment effect on centipede abundance.

Our data add to a growing body of evidence suggesting that
distantly related predators may engage in competitive interactions that
potentially limit population sizes of more basal consumers (Mokany and
Shine, 2003; Huntzinger and Karban, 2008; Jennings et al., 2010). These
studies do not imply that competition between related species is
unimportant, but they do suggest that competition among unrelated
species may be much more widespread than previously thought. Clearly,
unrelated pairs of species are more common in any ecosystem than are
related species, thus undetected interactions among these species may
have important roles in the determination of community and food web
structure. In our study, the removal of either salamanders or centipedes
resulted in significant changes in the abundance of other invertebrate
predators (i.e., spiders and carabid beetles) within the forest-floor
food web. We detected complex interactions that often involve multiple
groups such that removals positively affect some taxa but negatively
affect others. Our ability to test more specific hypotheses is limited
by the types of removals conducted, but in light of previous pair-wise
studies of salamanders and large predatory invertebrates (Gall et al.,
2003; Hickerson et al., 2004; Anthony et al., 2007; Figura, 2007), we
posit that interference competition rather than predator-prey
interactions (IGP) may be the best explanation for our findings. Our
data indicate that both direct (2 predator), and indirect (3 predator)
effects are operating in this system. For example, the negative
relationship between salamander abundance and spider abundance may be
the result of a direct interaction between salamanders and spiders.
Alternatively, the removal of centipedes may have had a direct positive
effect on salamander abundance and an indirect negative effect on spider
abundance. Dunham (2008) found that spiders were 2.3 times more abundant
in bird and mammal exclosures compared to control exclosures, a result
that is similar to ours. We agree with Dunham who points out the
difficulty in discerning whether the observed increase in spider
abundance in her predator exclosures was the result of reduced predation
by birds and mammals (a direct link), or reduced interspecific
competition in the absence of predators for macro-invertebrate prey.

We found a significant increase in the number of salamanders and
carabid beetles in centipede removal (CR) plots compared to controls.
Although the effectiveness of centipede removal was not detectable by
the next sampling date, the immediate and short term response by guild
members to each removal must have been sufficient to drive the
significant differences that we observed. It is impossible, however, to
say whether the changes in abundance were the result of direct
interactions, in which the removal of centipedes led to increases in
beetle abundance, or of indirect interactions, in which the removal of
centipedes led to increased numbers of salamanders, and in turn
increased numbers of beetles. The existing evidence on
beetle--salamander interactions indicates that the salamander, Plethodon
cinereus, and the carabid beetle, Platynus tenuicollis, are mutually
territorial (Gall et al., 2003); and, therefore, it is unlikely that
salamanders and carabid beetles would be positively associated with each
other. It is possible that in our CR treatment the temporary reduction
of centipedes between sampling dates was enough to elicit an increase in
carabid beetle abundance. However, we do not know whether centipedes
prey upon adult carabid beetles or whether they experienced competitive
release in the CR treatment. We currently are conducting laboratory
trials to determine which of these interactions is most likely
responsible for the observed numerical response. Preliminary analyses of
co-occurrence of carabid beetles and centipedes from the control
treatment ACOs indicate that these predators rarely occupy the same ACOs
at the same time.

Data on gut contents of predatory invertebrates like centipedes,
spiders, and beetles are few. However, data on stomach contents of
Plethodon cinereus reveal that spiders, centipedes, and carabid beetles
do not make up a significant proportion of the diet (Jaeger, 1990;
Maglia, 1996; Adams and Rohlf, 2000; Anthony et al., 2008). For example,
Maglia (1996) reported that spiders made up only 1.6% of the total
invertebrates by number in the diet of 172 P. cinereus from Tennessee,
and Stuczka (2011) found that centipedes, spiders, and beetles combined
made up only 3.2% of 4629 prey items taken from 258 salamanders at our
field site. Studies on asymmetrical IGP and predator diets, like those
described above, provide further support for competition, rather than
direct consumption, as the mechanism driving changes in the abundance of
predators in this forest-floor guild.

Recently there has been discussion about food web stability
occurring through fast and slow energy channels that are linked by
predators. Mobile predators couple strong and weak interaction chains by
switching between chains based on prey density (Rooney et al., 2006). In
forest ecosystems that experience alternating periods of moisture and
drying, species that have strict moisture requirements (amphibians and
arthropods) may move from one energy channel to another during wet
periods but may be restricted from doing so during dry periods between
rains. Plethodon cinereus, a terrestrial lungless salamander, can become
confined to moist, relatively simple environments and so the localized
effects of this predator should be most pronounced during dry periods
when prey are trapped within territories under cover objects. We found
that, in summer, interaction strengths between salamanders and IG
predators were of greater magnitude than in the spring and fall (Fig.
3b, points farthest from the zero line) suggesting that competitive
effects are strongest when salamanders are confined to microhabitat
beneath cover objects. During wetter periods, in the spring and fall,
when salamanders forage widely in the complex microhabitat of the
surrounding leaf litter and vegetation, their role may be more like that
described by Rooney et al. (2006) where predators regulate prey in fast
chains before moving from those depleted chains to the chains that have
experienced some degree of recovery. One interesting avenue of further
investigation would be to assess the direct effects of microhabitat
heterogeneity and moisture on top-down trophic cascades in the field by
examining relative abundances of these predator groups in structurally
complex versus simple habitats along a gradient from moist to dry.

A recent review of the literature discussing population regulation
and intraguild interactions in plethodontid salamanders concludes that
although we have learned a great deal in the past few decades concerning
interactions within guilds of salamanders, much remains to be explored.
Especially important in adding to our understanding of population and
community regulation are long-term, time series studies in unenclosed
field sites (Bruce, 2008). We examined interactions among top predators
within a forest-floor food web in a natural and unenclosed field
experiment focused on structurally simple microhabitats that may serve
as territories for forest-floor predators. We suggest that trophic
cascades may be localized in space and in time and we predict that
territorial defenders are more likely to generate strong top-down
effects than are widely foraging species.

Acknowledgments.--CMH was supported by a National Science
Foundation Doctoral Dissertation Improvement Grant (DEB-0608239). CDA
was supported by a John Carroll University Summer Faculty Fellowship and
by a George Grauel Faculty Fellowship. We thank J. Keiper and The
Cleveland Museum of Natural History for funding. This manuscript was
improved by the constructive comments of two anonymous reviewers. The
Cuyahoga Valley National Park granted permission to conduct fieldwork
and provided access to the Woodlake Environmental Field Station. Field
work was conducted under National Park Service scientific research
permit number CUVA-2004-SCI-0010 and IACUC protocol numbers JCU503 (JCU)
and 2604-WAL-AS (CSU).